NMR trial models: experiences with the colicin immunity protein Im7 and the p85α C-terminal SH2–peptide complex

Abstract

Two cases of successful molecular replacement using NMR trial models are presented. One is the crystal structure of the Escherichia coli colicin immunity protein Im7; the other is a heretofore unreported crystal structure of a speci®c PDGF receptor-derived peptide complex of the carboxy-terminal SH2 domain from the p85 subunit of human phosphatidylinositol 3-OH kinase. In both cases, molecular replacement was non-trivial. Success was achieved using trial models that consisted of an ensemble of NMR structures from which the more¯exible portions had been excized. Use of maximumlikelihood re®nement proved critical to be able to re®ne the poor starting models. The challenges typical of the use of NMR trial models in molecular replacement are discussed.

Tyrosine Phosphorylation of the Gα-Interacting Protein GIV Promotes Activation of Phosphoinositide 3-Kinase During Cell Migration

“…To gain insights into how these non-canonical phosphopeptides of GIV interact with the SH2 domains of p85α. we created the homology models ( Fig 4D, E; Fig S6 ) with the Internal Coordinate Mechanics (ICM) software (22, 23) using the crystal structures of the C-terminal SH2 domain of p85 in complex with pTyr 751 peptide from PDGFRβ (24), or the N-terminal SH2 domain of p85 bound to c-Kit phosphotyrosyl peptide (25) as templates. pTyr 1764 of GIV makes hydrogen bonds with residues of the C-terminal SH2 in a manner similar to the established crystal structure of this domain with pTyr 751 of PDGFRβ.…”

“…The initial coordinates for C-terminal and N-terminal SH2-domains of p85α were taken from their crystal structures bound to the PDGFRβ peptide, pY 751 VPML (24) and c-Kit phosphotyrosyl peptide (25), respectively. Structural models of GIV's phosphotyrosine peptides, EDTpY 1764 FISS and SNPpY 1798 ATLP were initially generated with ideal covalent geometry.…”

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins

“…Several cycles of manual model rebuilding and refinement were performed using Coot (37) and PHENIX (38), respectively. The structure of cSH2-OctP was determined as described above by using the structure of the cSH2 complexed with PDGFRderived peptide (Protein Data Bank code 1H9O) as a search model (39). The partially refined structure of cSH2-OctP was used as a search model for nSH2-OctP.…”

Somatic mutations in PI3Kα: Structural basis for enzyme activation and drug design

“…The structures of the N- and C-terminal SH2 domains bound to phosphopeptides revealed the structural basis for both phosphopeptide specificity and for the preference of a methionine residue at the fourth position of the motif Tyr-Xaa-Xaa-Met [20, 21]. In 1999, the first structure of a catalytic subunit of a PI3K enzyme became available when Williams and coworkers published the structure of four domains of the class 1b p110γ (RBD, C2, Helical and Kinase) [22].…”

“…Scheme of the domain structure of the heterodimer of class 1a. The structure of all domains of p85α have been determined individually (shown in patterned colors) (SH3: 1PNJ, 2PNI[43]; GAP: 1PBW[18]; nSH2: 2PNA,2PNB[19, 43], 2IUG, 2IUH, 2IUI[20],1OO4[44]; iSH2: 2V1Y[30]; cSH2: 1H90[21],1QAD[45],1BFI[46], 1PIC[47]); although only nSH2 and iSH2 domain structures(solid colors) have been determined as part of a complex with p110α [23, 24]. B. Ribbon diagram of the known structure of the p110α/niSH2 structure.…”